Electroporation is a technique for introducing macromolecules, which cannot penetrate the cell membrane, into cells using electric pulses. Electroporation is a widely used, and strongly recommended, method for cellular experiments and gene therapy. When a high electric field is applied, the cell membrane becomes temporarily porous and permeable to foreign materials, such as macromolecules. The electropermeabilization of the membrane depends on various parameters of the electric field, such as pulse width, pulse duration, the number of pulses, as well as other experimental conditions. Studies have been performed regarding the above-mentioned parameters in order to understand the mechanism of the electroporation which promotes the effect of transfection. For example, the magnitude of the electric field is reported to be an important parameter for increasing the permeability of the membrane and for controlling the portion of the cell membrane across which transmission occurs.
There are various devices for performing electroporation. FIG. 1 is a perspective view of a prior art cuvette that can be used for performing electroporation. FIG. 2 shows an electroporation device and system described in related U.S. application Ser. No. 10/560,301. FIG. 3 is a side view of the electroporation device shown in FIG. 2. FIG. 4 is a side view illustrating the components of a pipette tip used with the device shown in FIGS. 2 & 3.
An electric field can be applied to a cell suspension and/or gene mixture using the cuvette illustrated in FIG. 1. In such a system, the cuvette may be equipped with two parallel electrode plates 1. When a high electric field is applied between the two electrode plates, the cell membrane becomes permeable, making it is possible to introduce a gene or other foreign matter into the cell. Aluminum electrodes are cheap and may be used with a disposable cuvette. However, using Al creates Al3+ions dissolved from the aluminum electrodes, which ions have been found to negatively influence the cells. Furthermore, when aluminum electrodes are used, the magnitude of the electric field may vary due to a drop in electric voltage caused by the build up of oxide layers on the electrodes. Therefore, to generate a more constant electric field, it may be preferable to use platinum or gold electrodes. However, electrodes made from these materials are costly and therefore it is impractical to use electrodes made from these materials for consumable goods.
Thus, current electroporation devices have the following disadvantages. First, using the cuvette for electroporation may be expensive depending on the electrode material used since there are two electrodes mounted on two opposite side of the cuvette, and since each electrode has a wide surface area. Additionally, it is recommended that the cuvettes be single use. However, many users conduct experiments several times using the same cuvette, thus creating a high possibility of occurrence of experimental error. Second, because the electrode material (Al) is reactive in solution, and the overpotential relative to hydrogen generation is low, performing electroporation using current electroporation devices creates air bubbles due to decomposition of water on the surfaces of the electrodes. Third, the generated ion (Al3+) byproduct has been found to have a negative effect on cells. Fourth, surface resistance on the electrode is markedly increased due to the generation of an oxide layer (Al2O3) on the electrode surface. Fifth, the electric field between electrodes may not remain constant. This may be due to large quantities of current flowing through corners of the electrodes, thereby distorting the electric field. Finally, the sample volume needed may be large, which may make it difficult to analyze a small quantity of cells in the sample. Therefore, there is a need to develop a new electroporation device to address these disadvantages.
FIGS. 2-4 illustrate prior art versions of an electroporation device, pipette tip, and detailed components thereof for solving the above disadvantages. Referring to FIGS. 2-4, current electroporation devices may include a pulse generator 10 for generating an electric pulse; a sample reservoir 20; a pipette 30 with one side in electrical communication with the pulse generator; and a pipette tip 40, one end of which may be inserted onto the end portion of the pipette 30 and the other end of which may be inserted into the sample reservoir 20 and may be in fluid communication with the sample. Additionally, the distal end of the pipette 30, or the pipette tip 40, may be in fluid communication with the sample and the pipette 30 may be capable of drawing a sample into the pipette tip 40. Additionally, an electrode 50 may be inserted into the sample reservoir 20 and may be in fluid communication with the sample. The outer circumferential surface of the pipette 30 comprises a contact body 31 formed with conductive materials, wherein the contact body 31 may be electrically connected with a plunger 42 located inside the pipette tip 40 as shown in FIG. 3. The pipette tip 40 comprises a tubular body 41 which has openings at both ends, wherein the opening at one end may be a ring-shaped section having a relatively large diameter so as to be inserted into the end portion of the pipette 30, and the other end may be a ring-shaped section having a relatively small diameter. The pipette tip 30 is then inserted into the sample reservoir 20, and the plunger 42 located through body 41 of the pipette tip 40.
In performing electroporation using the system and electroporation device shown in FIGS. 2-4, the pipette draws the sample into the interior of the pipette tip 40 while maintaining pressure and thereby keeping the sample within the pipette tip 40. The sample reservoir 20 can then be replaced with a reservoir containing an electrolytic solution. By applying an electric current to the plungers 42 located within the body 41 of the pipette tip 40 and to the electrode 50 contacting the sample in the sample reservoir 40 or the electrolytic solution in the electrolytic solution reservoir, a cell located in the sample drawn into the pipette tip 40 may be electroporated, by passing a current between the two electrodes and through any cells located between the two electrodes. The electroporation of the cells located in the sample within the pipette tip 40 causes the cell membranes to become more permeable, and therefore facilitates the introduction of macromolecules, molecular probes, drugs, DNA, RNA, bacteria, genes, protein material, cells, or any other suitable substance which normally cannot penetrate the cell membrane, into the cell.
In the prior art electroporation system and device as shown in FIGS. 2-4, the outer circumferential surface of the pipette 30 comprises a separate contact body 31, and a separate plunger 42 which may be in electrical communication with the contact body 31 in the body 41 of the pipette tip 40. Since the outer circumferential surface of the pipette 30 includes a separate contact body 31 and a separate plunger 42 in electrical communication connecting with the contact body 31, the structure of the device may be complex which may make manufacturing the device both costly and time consuming. Since the contact body 31 and the plunger 42 formed on the outer circumferential surface of the pipette 30 are connected together in the interior of the device, it may be difficult for a user to determine the state of the connection between the plunger and the contact body.
Therefore, it would be useful to develop an electroporation device where the pipette tip itself can serve as an electrode and thereby apply and electric current to the sample without the need of a separate structure (such as plunger 42) serving as the electrode.